PROJECT SUMMARY Understanding the mechanisms behind pancreatic islet dysfunction in type 1 diabetics (T1D) would aid in the development of targetable therapies. Using a new approach to study the pancreas and isolated islets from individuals with recent-onset (<10 years duration) and longstanding T1D compared to controls, our lab is characterizing the type 1 diabetic islet in humans. We discovered that remaining T1D ? cells have nearly normal insulin secretion (relative to islet insulin content) in response to glucose, cAMP-evoked stimulation, and KCl-mediated depolarization. We showed by immunocytochemistry and qRT-PCR that remaining ? cells maintained their differentiation state (expression of PDX1, NKX6.1, NKX2.2). Unexpectedly, glucagon secretion by T1D ? cells was impaired and these cells had alterations in expression of transcription factors constituting ? cell identity (MAFB, ARX, NKX6.1). With decades of clinical evidence suggesting impaired counter regulation of blood glucose by glucagon in T1Ds, this proposal seeks to further explore the mechanisms behind ? cell dysfunction we have noted in T1D islets. We hypothesize that loss of ?-? cell contact and systemic hyperglycemia alters ? cell gene expression, leading to impaired glucagon secretion. To test this hypothesis, we propose to first determine whether loss of ?-? cell contact in normal human ? cells produces gene expression and functional changes. We will then determine whether systemic hyperglycemia exacerbates the effects of ? cell loss on ? cell identity and function. With transplantation of normal human ? cells into the anterior eye chamber of immunodeficient Nod-SCID-IL2R?null; RIP-DTR (NSG-DTR) mice where endogenous mouse ? cells can be ablated by a single diphtheria toxin (DT) injection, we will study the ? cell response under either normoglycemic or hyperglycemic conditions in vivo. Outcomes of this aim will determine if loss of ?-? cell contact and/or hyperglycemia are sufficient to trigger T1D ? cell associated molecular and functional changes in normal human ? cells. Secondly, we will define the molecular mechanism of impaired glucagon secretion to hypoglycemia by reconstituting the gene expression changes of the T1D ? cell in normal human ? cells and study calcium activity and cAMP response to epinephrine in hypoglycemic conditions. Aim 2 will determine whether molecular changes in the T1D ? cell lead to impaired epinephrine mediated cAMP and calcium signaling, and subsequently glucagon secretion. The proposed studies will clearly define the non- autoimmune contributors to an altered T1D ? cell phenotype, and determine how these gene expression changes lead to impaired glucagon secretion in the hypoglycemic state. This will help us further define the mechanism by which impaired glucagon secretion occurs in type 1 diabetics and develop new therapies to target these processes.